Single chain peptide compounds having hemoregulatory activity

ABSTRACT

There is disclosed single chain peptide compounds, substituted at a Cα-atom of a non-terminal amino acid by a group--A which is defined in claim 1. The native α-side chain of the Cα atom bonded group to group --A absent. The peptide derivatives according to the invention are useful for inhibiting cell proliferation, especially myelopoietic and bone marrow cells.

The present invention relates to the use of peptides having aninhibitory effect on cell proliferation, and to novel peptides havingspecific and/or general cell inhibitory effects.

The mammalian body contains cells having enormously diverse structuresand functions, and the mechanisms of differentiation and developmenthave been the focus of much study. It is known that for systems of cellshaving a continuous turnover the mechanism commonly involves a reservoirof pluripotent stem cells which divide and constantly supply new cellsto the system. While initially homogeneous the stem cells supplied fromthe "reservoir" soon become committed to one or other morphology andsubsequently develop into the required functional cells.

Examples of such stem cell systems are the haemopoietic system in bonemarrow and the epithelial and epidermal systems.

The manipulation or control of stem cell division is of great potentialtherapeutically and much research continues to be devoted to elucidatingthe mechanisms involved and the chemical messengers responsible. To dateseveral biomolecules have been identified as possessing a role in cellproduction and differentiation either by the stimulation or inhibitionof a step within the process. Myelopoiesis has been particularly wellstudied in this regard and molecules involved in its control include:colony-stimulating factors (CSF) such as granulocyte colony-stimulatingfactor (G-CSF), macrophage colony-stimulating factor (M-CSF),granulocyte-macrophage colony-stimulating factor (GM-CSF), multi-lineagecolony-stimulating factor (multi-CSF; IL-3) [see Metcalf, Science 229:16 (1985)], interleukin 11 (IL-11) [see Paul et al Proc Natl Acad SciUSA 87: 7521 (1990)], Lactoferrin [see Broxmeyer et al Blood Cells 11:429 (1986)], prostoglandins [see Pelus et al J. Immunol 140: 479(1988)], acidic (H-subunit) ferritin [see Broxmeyer et al Blood 68: 1257(1986)], interferons (α, β and γ) [see Pelus et al supra. and Broxmeyeret al J. Immunol 131: 1300 (1983)], tumor necosis factors (α and β) [seeBroxmeyer et al J Immunol 136: 4487 (1986)], transforming growthfactor-β [see Ottman et al J Immunol 140: 2661 (1988)], and activin andinhibin [see Broxmeyer et al Proc Natl Acad Sci USA 86: 779 (1989)].

It has also been found that the haemoregulatory pentapeptide(pGlu-Glu-Asp-Cys-Lys SEQ ID NO 1) inhibits the proliferation ofmyelopoietic cells selectively [see Paukovits et al Z. Naturforsch 37:1297 (1982)] and other peptides corresponding to a narrow generalformula were discovered to exert a similar inhibitory effect inhemopoiesis [see EP-A-112656 and WO90/02753]. Oxidation of the peptidemonomers resulted in dimeric molecules linked by a cysteine bridge andthese dimeric molecules were found to stimulate myelopoiesis [see Laerumet al. Exp. Hematol 16: 274 (1988)]. The (pGlu-Glu-Asp-Cys-Lys SEQ IS NO1)₂ dimer and other similar compounds are disclosed in WO-A-88/03535.Further dimeric peptide compounds are disclosed in EP-A-408371 in whichthe disulphide bond has been replaced by a carbon or carbon/sulphurbridge linking the selected peptide chains. The bridge is thusrelatively stable to hydrolysis but is itself inert and incapable ofparticipating in receptor-dimer interactions.

While we do not wish to be bound by theoretical considerations, it ispresently believed that such peptide compounds interact with stromalcells in vivo and that the stromal cells are responsible for stimulatingor inhibiting cellular division via other soluble factors. The dimersare thus believed to induce or promote stromatic production ofstimulatory cellular regulatory factor(s) whilst the monomeric peptidesmay either inhibit that process or cause the production of factors whichprevent or hinder cell division. Thus, according to current thinking,the stromal cells may act to amplify the stimulatory or inhibitoryeffects of the dimeric and monomeric peptides respectively.

There is a continuing need for peptide compounds capable of inhibitingcell proliferation usefully in vivo. In this regard it should be notedthat different degrees of inhibition may be more appropriate to certainclinical situations than to others and, in particular, selectiveinhibition of individual cell types is important.

The present invention provides a peptide derivative comprising asingle-chain hemoregulatory, for example haemopoesis-inhibiting, peptidewherein a Cα atom of a non-terminal amino acid thereof is substituted bya group --A, the native α-side chain being absent from said Cα atom,where A is --CR^(A') R^(A) --Z

where each R^(A) is independently a hydrogen atom or a group --R^(A"),--OR^(A"), --SR^(A"), --NR^(A") R^(A"), CONR^(A") R^(A") or --COOR^(A");

R^(A') is a hydrogen atom or a group R^(A") ;

R^(A") is an alkyl, cycloalkyl, alkanoyl, hydroxyalkyl, anidine group,or a carbocyclic or heterocyclic group;

Z is a group --OR^(B), --NR^(C) R^(C), --CR^(D) R^(E) R^(F) or asubstituted or unsubstituted, aromatic or non-aromatic, carbocyclic orheterocyclic ring other than unsubstituted phenyl;

wherein each R^(B) is a straight chained or branched, saturated orunsaturated hydrocarbon group such as an alkyl, aralkyl or aryl group,optionally substituted by one or more R^(A) groups, where R^(A) is asdefined above, and optionally interrupted by one or more --N--, --O-- or--S-- heteroatoms;

R^(C) is a hydrogen atom or a group R^(B) ;

R^(D) is a hydrogen atom or a group R^(F) ;

R^(E) is a hydrogen atom or a group R^(F), or together with group R^(D)forms a >C=0 group; and

R^(F) is a group --R^(B), --OR^(B), --NR^(C) R^(C) or --SR^(B), or ahydroxy, carboxy, aminocarbonyl or alkoxy group, or is a methylene grouplinked to the nitrogen atom attached to the Cα atom, or together withR^(D) forms an alkylidene group, or is a hydrogen atom where one ofR^(A), R^(A'), R^(D) and R^(E) is other than hydrogen.

Where any group R^(A) or R^(B) is a C-attached organic group itpreferably contains 1 to 10 carbon atoms, especially 1 to 6 carbonatoms. Alkyl groups may be straight chained or branched and may besubstituted by aryl groups having 6-10 carbon atoms (ie. forming anaralkyl group), alkoxy, hydroxy, acyloxy, amino, azido, acylamino,aminocarbonyl or carboxy groups. Aryl groups include 5- or 6-memberedheterocyclic aryl groups having one or more heteroatoms selected from O,N or S such as furyl, imidazolyl, pyrrolyl, pyridinyl and thienylgroups. Substituents which may be present on aryl groups include C₁₋₆alkyl groups, hydroxy and carboxy groups. Examples include methyl,ethyl, propyl, t-butyl, pentyl, carboxyethyl and benzyl groups.

In one embodiment, Z represents a substituted or unsubstituted, aromaticor non-aromatic, carbocyclic or heterocyclic ring other thanunsubstituted phenyl.

Particularly preferred groups for Z include benzyl, indolyl,hydroxyphenyl, imidazolyl, naphthyl, thienyl, pyridinyl, furanyl,isoxazolyl, 3,5-dimethylisoxazolyl or cyclohexyl groups.

In another embodiment Z represents a group --OR^(B) (where R^(B) is asdefined above) or --CHR^(D) R^(F) (where R^(D) represents a hydrogenatom or a group --CH₃ ; R^(F) represents an alkyl group optionallysubstituted by hydroxyl, amino or azide groups; or R^(D) and R^(F)together form an alkylidine group).

Preferably both R^(A) and R^(A') represent hydrogen so that --A is agroup --CH₂ --Z.

Any single chain peptide which exhibits a hemoregulatory effect issuitable as the peptide which is substituted in accordance with theinvention.

Alternatively expressed, the invention provides compounds according tothe present invention in which the said hemoregulatory peptide chaininclude those of formula: ##STR1## (wherein n and m independentlyrepresent 0 or 1; p, q and r independently represent 1 or 2;

s represents 3 or 4;

R¹ and R² are both hydrogen atoms or together represent an oxo group;

R³ and R⁴ are both hydrogen atoms or together represent a carbon-carbonbond;

R⁵ is hydrogen or an acyl group;

each R⁶ and R⁷ independently represent a hydroxy group or an aminogroup, but are preferably hydroxy groups,

R⁸ represents hydrogen; a C₂₋₆ alkyl group; a C₇₋₂₀ aralkyl group, whichmay carry one or more hydroxy, amino or methoxy substituents; or ametabolically labile S-protecting group;

R⁹ represents hydrogen or a methyl group; and

R¹⁰ represents a hydroxy or a substituted or unsubstituted amino group,the residue of the amino acid glutamine or a peptide having anN-terminal glutamine unit) and salts thereof.

All the said amino acid residues may be in either the D or the L form.The L-form of the amino acids is, however, preferred.

Where an N-terminal protecting group R⁵ is present this may, asindicated above, be an acyl group having 1-20 carbon atoms, e.g. a loweralkanoyl group having 1-5 carbon atoms such as the acetyl group, or anaroyl or aralkanoyl group having 7 to 20 carbon atoms such as thebenzoyl or phenylacetyl group.

R⁵ may also be an acyl group derived from an amino acid or a peptidechain. In particular, R⁵ may be an acyl group derived from serine or anyof the peptides derived from the following amino acid sequence byremoval of successive N-terminal amino acids:Lys-Ile-Ile-His-Glu-Asp-Gly-Tyr-Ser SEQ ID NO 2.

The terminal amino group of the overall peptide of formula (I) ispreferably protected, e.g. by acylation with an alkanoyl, aralkanoyl oraroyl group.

Where R⁸ is a C₂₋₆ alkyl group this may, for example, be an ethyl, butylor hexyl group. When R⁸ is an aralkyl group, this may conveniently be anarylmethyl group such as benzyl, diphenylmethyl or triphenylmethyl.Where R⁸ is a metabolically labile group this may, for example, be anarylthio group having 5 to 10 carbon atoms, e.g. the pyridyl thio group,or an acyl group as defined above.

The compounds of the invention are preferably pentapeptides, that is nis preferably 0.

The cyclic groups in the R^(a) residue are preferably five-membered,that is m is preferably 0.

Insofar as any of the peptides defined by formula I above are of low ornegligible haemoregulatory activity, they may nevertheless be effective,in the peptide compound derivative according to the invention, ininhibiting cell proliferation.

In peptide compound derivatives formed from a peptide chain as describedin formula I the substitution of the chain is desirably at R^(d).

Particularly preferred peptide compound derivatives according to thepresent invention are those of formula II ##STR2## wherein R^(a), R^(b),R^(c), R^(e), R^(f), A and n are all defined as above and the group--NH--CH--CO-- is the derivatized form of R^(d) which is attached togroup --A in such a manner that it's native side chain is absent. Theinvention also includes compounds of formula II in which A together withthe adjacent group --N--CH--CO-- form a proline residue.

One especially preferred peptide compound of formula II is ##STR3##where group --A is the group as discussed above.

The ability of the peptide derivatives of the invention to inhibitproliferation of a wide range of cells in addition to or even excludingthe haemopoietic system is of value in medicine either where excessivecell proliferation requires treatment, as in psoriasis, or where cancertherapy would be likely to damage a particular cell population. Manycell types are particularly susceptible to the cytotoxic drugs orradiations used in anti-cancer therapy and one known technique is to usea drug to inhibit proliferation of cells such as those of thehaemopoietic system during the anticancer therapy, followed byresumption of normal proliferation when the effect of the inhibitorydrug has disappeared. The peptides of the present invention appear tohave appropriately short biological half-lives for such therapy.Similarly, proliferation of selected populations of cells susceptible tocancer therapy may be inhibited together with the cancer cellsthemselves and the anti-cancer therapy is initiated only when the cancercells have reached a susceptible phase of proliferation while the normalcells are in a less susceptible phase.

One type of cell proliferation occurs when cells such as bone marrowcells, phagocytes or granulocytes are stimulated by CSF drugs duringtherapy. Inhibition of cell growth can restore such cells to normalgrowth rates.

In many autoimmune diseases, the subject produces leucocytes activeagainst their own tissues. By inhibiting leucocyte function, at leastfor a time, such autoimmune reactions may be correspondingly reduced.

Another clinical application will be in combination with thecorresponding dimers or related myelopoiesis stimulators as disclosed inWO-A-88/03535 to induce alternating peaks of high and low activity inthe bone marrow cells, thus augmenting the natural circadian rhythm ofhaemopoiesis. In this way, cytostatic therapy can be given at periods oflow bone marrow activity, thus reducing the risk of bone marrow damage,while regeneration will be promoted by the succeeding peak of activity.

In general, in order to exert an inhibitory effect, the peptides of theinvention may be administered to human patients orally or by injectionin the dose range 0.001-100 mg, for example 1-5 mg, per 70 kg bodyweight per day. If administered intravenously or subcutaneously, thedose may be in the range 1-10 mg per 70 kg body weight per day, forexample about 6 mg, for up to ten days. Nasal, topical (transdermal) orrectal administration is, of course, also feasible. In principle it isdesirable to produce a concentration of the peptide of about 10⁻¹³ M to10⁻⁵ M in the extracellular fluid of the patient.

According to a further feature of the present invention there areprovided pharmaceutical compositions comprising as active ingredient oneor more peptide compound derivatives as hereinbefore defined orphysiologically compatible salts thereof, in association with apharmaceutical carrier or excipient. The compositions according to theinvention may be presented, for example, in a form suitable for oral,nasal, parenteral or rectal administration.

As used herein, the term "pharmaceutical" includes veterinaryapplications of the invention.

The compounds according to the invention may be presented in theconventional pharmacological forms of administration, such as tablets,coated tablets, nasal sprays, solutions, emulsions, powders, capsules orsustained release forms. Conventional pharmaceutical excipients as wellas the usual methods of production may be employed for the preparationof these forms. Tablets may be produced, for example, by mixing theactive ingredient or ingredients with known excipients, such as forexample with diluents, such as calcium carbonate, calcium phosphate orlactose, disintegrants such as corn starch or alginic acid, binders suchas starch or gelatin, lubricants such as magnesium stearate or talcum,and/or agents for obtaining sustained release, such ascarboxypolymethylene, carboxymethyl cellulose, cellulose acetatephthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets maybe produced by coating cores, obtained in a similar manner to thetablets, with agents commonly used for tablet coatings, for example,polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxideor sugar. In order to obtain sustained release or to avoidincompatibilities, the core may consist of several layers too. Thetablet-coat may also consist of several layers in order to obtainsustained release, in which case the excipients mentioned above fortablets may be used.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers either with an aerosol propellant or provided withmeans for manual compression. Capsules containing one or several activeingredients may be produced, for example, by mixing the activeingredients with inert carriers, such as lactose or sorbitol, andfilling the mixture into gelatin capsules.

Suitable suppositories may, for example, be produced by mixing theactive ingredient or active ingredient combinations with theconventional carriers envisaged for this purpose, such as natural fatsor polyethyleneglycol or derivatives thereof.

Dosage units containing the compounds of this invention preferablycontain 0.1-10 mg, for example 1-5 mg of the peptide of formula (I) orsalt thereof.

The present invention provides the peptide compounds and compositionsdescribed above for use in the inhibition of cell division, especiallymyelopoiesis and bone marrow regeneration. Use of the peptide compoundsaccording to the invention in the manufacture of a medicament to inhibitcell division such as myelopoiesis and bone marrow regeneration alsoforms a further aspect of this invention.

According to a still further feature of the present invention there isprovided a method of inhibition of cell division, especiallymyelopoiesis which comprises administering an effective amount of acompound or a pharmaceutical composition as hereinbefore defined to asubject.

A further major use of the new peptide compound derivatives, however, isin the production of material for immunological assay techniques. Thepeptide may then be covalently attached to a suitable high molecularcarrier such as albumin, polylysine or polyproline in order to beinjected into antibody-producing animals (e.g. rabbits, guinea pigs orgoats). In vitro immunisation techniques may also be used. Highspecificity antisera are obtained by use of well known absorptiontechniques, using the high molecular carrier. By introducingradioactivity (³ H, ¹²⁵ I, ¹⁴ C, ³⁵ S) into the peptide molecule, aradioimmuno assay can be designed and used for determining the peptidein the different biological fluids such as serum (plasma), urine andcerebrospinal fluid.

The peptides of the invention may be synthesised in any convenient way.Suitable methods for forming the amino acid units are described in, forexample, "Synthesis of Optically Active α-Amino Acids" by Robert M.Williams (Pergamon Press, 1989). In general, the reactive side chaingroups present (amino, thiol and/or carboxyl) will be protected duringthe coupling reactions of the overall synthesis but it is possible toleave some side chain groups unprotected (hydroxy groups, imidazolegroups, primary amide groups, amide groups in cyclic amino acids likepyroGlu) during the entire synthetic procedure.

The final step will thus be the deprotection of a fully protected or apartly protected derivative of a peptide of the general formula I andsuch processes form a further aspect of the invention.

Schollkopf et al have described the preparation of a variety of aminoacids by the metallation and subsequent alkylation of bis-lactim ethers(see, for example, Tetrahedron 39: 2085 (1983) and Topics Curr Chem 109:65 (1983)). An adaptation of this method has proved particularly usefulfor the preparation of the substituted amino acids which form the basisof the present invention. In particular, a bis-lactim ether derived froma valine-glycine dipeptide forms a useful starting compound for thesubstitution reaction which may be summarized as follows: ##STR4##(wherein X is a leaving group, for example a bromine atom).

(S) α-amino acid derivatives can be prepared by this method if D-valineis initially used to form the bis-lactim ether. Equally, (R)-α-aminoacid derivatives may be formed by the use of L-valine.

Thus, the present invention also provides a process for producing apeptide compound according to the invention comprising deprotecting apartially or fully protected derivative thereof.

The present invention further provides a process for producing a peptidecompound according to the invention, comprising

a) metallating and subsequently alkylating a bis-lactim ether to form abis-lactim dipeptide ether;

b) hydrolysing a bis-lactim dipeptide ether of step (a) to form an aminoacid of formula NH₂ --CH(A)--COOH wherein A is as defined above;

c) introducing the remaining amino acids in the peptide chain; and

d) deprotecting any protected group.

The substituted bis-lactim dipeptide ethers and substituted α-amino acidproduced by this technique form a further aspect of the presentinvention.

Additionally, the invention also covers amino-protected, hydroxylprotected, thiol protected and/or carboxyl protected peptide derivativesof the invention.

Once the substituted α-amino acid has been formed, then the remainingamino acids in the peptide chain can be introduced using conventionaltechniques.

In building up the peptide chains, one can in principle start either atthe C-terminal or the N-terminal although only the C-terminal startingprocedure is in common use.

Thus, one can start at the C-terminal by reaction of a suitablyprotected derivative of the first amino acid with a suitably protectedderivative of the second amino acid. The first amino acid derivativewill have a free α-amino group while the other reactant will have eithera free or activated carboxyl group and a protected amino group. Aftercoupling, the intermediate may be purified for example bychromatography, and then selectively N-deprotected to permit addition ofa further N-protected and free or activated amino acid residue. Thisprocedure is continued until the required amino acid sequence iscompleted.

Carboxylic acid activating substituents which may, for example, beemployed include symmetrical or mixed anhydrides, or activated esterssuch as for example p-nitrophenyl ester, 2,4,5,trichlorophenylester,N-hydroxybenzotriazole ester (OBt), N-hydroxy-succinimidylester (OSu) orpentafluorophenylester (OPFP).

The coupling of free amino and carboxyl groups may, for example, beeffected using dicyclohexylcarbodiimide (DCC). Another coupling agentwhich may, for example, be employed isN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

In general it is convenient to effect the coupling reactions at lowtemperatures, for example, -20° C. up to ambient temperature,conveniently in a suitable solvent system, for example, tetrahydrofuran,dioxan, dimethylformamide, methylene chloride or a mixture of thesesolvents.

It may be more convenient to carry out the synthesis on a solid phaseresin support. Chloromethylated polystyrene (cross-linked with 1%divinyl benzene) is one useful type of support; in this case thesynthesis will start the C-terminal, for example by coupling N-protectedlysine to the support.

A number of suitable solid phase techniques are described by EricAtherton, Christopher J. Logan, and Robert C. Sheppard, J. Chem. Soc.Perkin I, 538-46 (1981); James P. Tam, Foe S. Tjoeng, and R. B,Merrifield J. Am. Chem. Soc. 102, 6117-27 (1980); James P. Tam, RichardD. Dimarchi and R. B. Merrifield Int. J. Peptide Protein Res 16 412-25(1980); Manfred Mutter and Dieter Bellof, Helvetica Chimica Acta 672009-16 (1984).

It is also possible for the coupling reactions to be performed insolution.

A wide choice of protecting groups for amino acids are known and areexemplified in Schroder, E., and Lubke, K., The Peptides, Vols. 1 and 2,Academic Press, New York and London, 1965 and 1966; Pettit, G. R.,Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970,1971, 1975 and 1976; Houben-Weyl, Methoden der Organischen Chemie,Synthese von Peptiden, Band 15, Georg Thieme Verlag Stuttgart, NY, 1983;The Peptides, Analysis, synthesis, biology 1-7, Ed: Erhard Gross,Johannes Meienhofer, Academic Press, NY, San Fransisco, London; Solidphase peptide synthesis 2nd ed., John M. Stewart, Janis D. Young, PierceChemical Company.

Thus, for example amine protecting groups which may be employed includeprotecting groups such as carbobenzoxy (Z-), t-butoxycarbonyl (Boc-),4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr-), and9-fluorenylmethoxycarbonyl (Fmoc-). It will be appreciated that when thepeptide is built up from the C-terminal end, an amine protecting groupwill be present on the α-amino group of each new residue added and willneed to be removed selectively prior to the next coupling step. Forsolid phase systems one particularly useful group for such temporaryamine protection is the Fmoc group which can be removed selectively bytreatment with piperidine in an organic solvent. For synthesis insolution, Boc- is a preferred protecting group, which can be introducedand removed in a conventional manner.

The amino acids or peptides often require to be silylated prior toprotection eg. by addition of Fmoc in order to improve their solubilityin organic solvents. Silylation and Fmoc protection reactions aresummarized below: ##STR5## Carboxyl protecting groups which may, forexample be employed include readily cleaved ester groups such as benzyl(--OBZl), p-nitrobenzyl (--ONB), or t-butyl (--tOBu) as well as thecoupling on solid supports, for example methyl groups linked topolystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) andacetamidomethyl (Acm).

It will be appreciated that a wide range of other such groups exists as,for example, detailed in the above-mentioned literature references, andthe use of all such groups in the hereinbefore described processes fallwithin the scope of the present invention.

A wide range of procedures exists for removing amine- andcarboxyl-protecting groups. These must, however, be consistent with thesynthetic strategy employed. The side chain protecting groups must bestable to the conditions used to remove the temporary α-amino protectinggroups prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups suchas tOBu may be removed simultaneously by acid treatment, for examplewith trifluoro acetic acid. Thiol protecting groups such as Trt may beremoved selectively using an oxidation agent such as iodine.

The cysteine containing peptides may be synthesised by the methodsdescribed in the text with removal of all protecting groups includingthe thiol protecting groups as the last synthetic step.

The following Examples are given by way of illustration only.

EXAMPLE 1(S)-2-(9-Fluorenylmethoxycarbonylamino)-5-phenyl-4-(E)-penten-1-oic-acid

(S)-2-Amino-5-phenyl-4-(E)-penten-1-oic acid was prepared from(R)-(-)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine and cinnamylbromide by analogy to the procedure described by U. Schollkopf, U. Grothand C. Deng in Angew. Chem. 93 (1981) 793. The amino acid (4.0 mmol)thus obtained, is added to a mixture of hexamethyldisilazane (15 ml) andtrimethylchlorosilane (2.0 ml), the mixture heated at 100° C. withstirring under N₂ overnight. Excess silylating reagent is then distilledoff, the residue dissolved in CH₂ Cl₂ (10 ml), a solution of9-fluorenylmethoxycarbonyl chloride (4.1 mmol) in dry CH₂ Cl₂ (5 ml)added, the mixture stirred at ambient temperature under N₂ for 3 hours,the CH₂ Cl₂ evaporated, the residue dissolved in THF (9 ml) and water (1ml), the solution stirred for 30 minutes, evaporated, the residuedissolved in EtOAc, dried (MgSO₄) and evaporated to yield the titlecompound for the subsequent peptide synthesis.

EXAMPLE 2(2S,4R,5R)-N-9-Fluorenylmethyloxycarbonyl-N'-benzyloxycarbonyl-4,5-dihydroxy-4,5-O,O-isopropylidenelysine

a)(2R,3R)-1-Bromo-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-2,3-dihydroxy-2,3-O,O-isopropylidene-butane

(2R)-2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (3.24 g, 17.36 mmol)was dissolved in 60 ml anhydrous THF and the solution was cooled to -78°C. A solution of BuLi in hexane (10.85 ml, 17.36 mmol) was added and thesolution was stirred for 30 minutes. After addition of 7.8 ml1,3-dimethyl-2-imidazolidinone stirring was continued for an additional15 minutes. A solution of(2R,3R)-1,4-dibromo-2,3-dihydroxy-2,3-O,O-isopropylidenebutane (5.0 g,17.36 mmol) in 10 ml THF was added dropwise and the solution came toambient temperature overnight. After hydrolysis with phosphate buffer(pH 7) the reaction mixture was extracted several times with diethylether, the organic solutions dried (MgSO₄), concentrated and the residuepurified by flash chromatography (hexane/ethyl acetate 9/1).

Yield: 3.00 g (44.1%).

¹ H NMR (CDCl₃) δ: 0.7(d,3H), 1.04 (d,3H), 1.40 (s,3H), 1.41 (s,3H),1.95-2.30 (m,3H), 3.40-3.58 (m,2H), 3.68 (s,3H), 3.70 (s,3H), 3.97(dd,1H), 4.03-4.20 (m,3H).

¹³ C NMR (CDCl₃) δ: 16.74, 19.03, 27.29, 27.46, 31.88, 32.66, 37.83,52.40, 52.42, 52.55, 60.76, 79.80, 109.22, 163.21, 164.00 FAB-MS signalat m/z 391.2 (72), 141.1 (100).

b)(2R,3R)-1-Azido-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-2,3-dihydroxy-2,3-O,O-isopropylidene-butane

(2R,3R)-1-Bromo-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl)-2,3-dihydroxy-2,3-isopropylidenebutane(5.51 g, 14.11 mmol) were dissolved in 50 ml DMF and 2.93 g NaN₃ (45mmol) and 0.1 g NBu₄ I were added. The solution was kept between 70° and80° C. overnight and diluted with 50 ml water. After extraction withchloroform the organic layers were collected and dried (MgSO₄). Theresidue was purified by flash chromatography (hexane/ethyl acetate 7/3).

Yield: 4.06 g (81.5%).

¹ H NMR (CDCl₃) δ: 0.68 (d,3H), 1.03 (d,3H), 1.40 (s,3H), 1.42 (s,3H),1.95-2.30 (m,3H), 3.20-3.80 (m,2H), 3.65 (s,3H), 3.68 (s,3H), 3.95(dd,1H), 4.00-4.15 (m,3H).

¹³ C NMR (CDCl₃) δ: 16.72, 19.02, 26.92, 27.27, 31.85, 37.52, 52.10,52.36, 52.41, 52.47, 60.70, 75.09, 79.64, 109.13, 163.17, 164.00 FAB-MSsignal at m/z 354.3 (100), 141.1 (75).

c)(2R,3R)-1-Benzyloxycarbonylamino-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-2,3-dihydroxy-2,3-O,O-isopropylidene-butane

(2R,3R)-1-Azido-4-((2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl)-2,3-dihydroxy-2,3-O,O-isopropylidenebutane(3.57 g, 10.12 mmol) was dissolved in 60 ml EtOH and 1.3 g Pd/C wereadded. The solution was purged with argon. Hydrogen was bubbled throughthe solution and it was kept under hydrogen overnight and filteredthrough celite. The residue was dried under vacuum and dissolved in 30ml dioxane. 15 ml 1M NaHCO₃ solution (15 mmol) were added and 1.84 mlCbzCl (13 mmol) were added at 0° C. The solution was stirred at thistemperature for 1 hour, diluted with water and extracted withchloroform. The residue was purified by flash chromatography(hexane/ethyl acetate 8/2).

Yield: 2.20 g (44.1%).

¹ H NMR (CDCl₃) δ: 0.67 (d,3H), 1.03 (d,3H), 1.35 (s, 3H), 1.38 (s,3H),1.90-2.05 (m,1H), 2.10-2.30 (m,2H), 3.30-3.55 (m,2H), 3.64 (s,3H), 3.68(s,3H), 3.92-4.15 (m,4H), 5.09 (s,2H), 5.45 (m,1H), 7.26-7.38 (m,5H). ¹³C NMR (CDCl₃) δ: 16.70, 19.05, 27.17, 31.83, 37.69, 42.94, 52.26, 52.41,52.52, 60.65, 66.72, 75.69, 79.41, 108.61, 128.02, 128.07, 128.47,136.54, 156.40, 163.24, 164.11 FAB-MS signal at m/z 462.3 (56), 141.1(47), 91.0 (100) C₂₄ H₃₅ N₃ O₆ (461.6): Calc.: C, 62.45; H, 7.64; N,9.10 Found: C, 62.99; H, 7.67; N, 8.77

d)(2S,4R,5R)-N'-Benzyloxycarbonyl-4,5-dihydroxy-4,5-O,O-isopropylidene-lysinemethyl ester

(2R,3R)-1-Benzyloxycarbonylamino-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-2,3-dihydroxy-2,3-O,O-isopropylidenebutane(0.5 g, 1.08 mmol) was dissolved in 5 ml dioxane and 4.20 ml 0.5M HCl(2.10 mmol) were added dropwise and stirred under argon at ambienttemperature for 5 hours. Ammonia solution was added until pH 9 wasreached, and the solution was extracted with chloroform, dried (MgSO₄),and the valine methyl ester was removed by bulb-to-bulb distillation(0.05 Torr, 25° C.). Yield; 0.40 g (100%).

¹ H NMR (CDCl₃) δ: 1.35 (s,6H), 1.70-2.20 (m,4H), 3.20-3.60 (m,3H), 3.70(s,3H), 3.73-3.90 (m,2H), 5.09 (s,2H), 5.32 (m, 1H), 7.26-7.40 (m, 5H).¹³ C NMR (CDCl₃) δ: 27.01, 27.05, 37.56, 42.11, 51.99, 52.38, 66.79,75.79, 79.82, 108.99, 128.00, 128.44, 136.39, 156.46, 175.30

(2S,4R,5R)-N'-Benzyloxycarbonyl-4,5-dihydroxy-4,5-O,O-isopropylidene-lysine

(2S,4R,5R)-N'-Benzyloxycarbonyl-4,5-dihydroxy-4,5-O,O-isopropylidenelysinemethyl ester (0.40 g , 1.08 mmol) was dissolved in 3.5 ml water and 4 mldioxane and cooled to 0° C. 0.54 ml 2N LiOH solution (1.08 mmol) wereadded, and the solution was stirred under argon overnight. TLC controlshowed quantitative formation of(2S,4R,5R)-N'-benzyloxycarbonyl-4,5-dihydroxy-4,5-isopropylidenelysine.This crude solution was directly used for the next step.

(2S,4R,5R)-N-9-Fluorenylmethyloxycarbonyl-N'-benzyloxycarbonyl-4,5-dihydroxy-4,5-O,O-isopropylidene-lysine

2.5 ml 1M NaOH solution (2.5 mmol) and a solution of 0.518 g9-fluorenyloxycarbonylchloride (2.00 mmol) in 3 ml dioxane were added toa crude solution of(2S,4R,5R)-N'-benzyloxycarbonyl-4,5-dihydroxy-4,5-isopropylidenelysineand the mixture stirred overnight. The solution was acidified to pH 2 byaddition of a KHSO₄ -solution, extracted with ethyl acetate, dried(MgSO₄), and the residue was purified by flash chromatography(chloroform/MeOH 8/2).

Yield: 310 mg (50.0%).

¹ H NMR (DMSO/D₂ O) δ: 1.19 (s,3H), 1.22 (s,3H), 1.60-2.10 (m,2H),2.90-3.30 (m,2H), 3.50-4.00 (m, 3H), 4.00-4.40 (m,3H), 4.95 (s,2H),7.20-8.00 (m,13H). ¹³ C NMR (DMSO/D₂ O) δ: 27.59, 27.81, 37.14, 43.15,53.94, 65.92, 66.01, 76.98, 80.16, 108.40, 120.61, 125.67, 125.79,127.66, 128.16, 128.22, 128.32, 128.89, 137.55, 141.18, 144.36, 156.14,156.87, 176.09 FAB-MS signal at m/z 497.1 (3), 429.1 (4), 179.1 (31),91.0 (64) C₃₂ H₃₄ N₂ O₈ (574.6): Calc.: C, 66.89; H, 5.96; N, 4.88Found: C, 62.53; H, 5.60; N, 5.03

EXAMPLE 3 (S)-2-(9-Fluorenylmethyloxycarbonylamino)-6-azidohexanoic acid

a)1-Bromo-4-[(2R,5)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-butane

(2R)-2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (3.76 g, 20.43 mmol)was dissolved in 60 ml anhydrous THF and the solution was cooled to -78°C. A solution of nBuLi in hexane (20.43 mmol, 12.77 ml) was added andthe solution was stirred for 30 minutes. A solution of 1,4-dibromobutane(4.41 g, 20.43 mmol) in 10 ml THF was added dropwise and the solutioncame to ambient temperature overnight. After hydrolysis with phosphatebuffer (pH 7) the reaction mixture was extracted several times withdiethyl ether, the organic solution dried (MgSO₄) and the residue waspurified by flash chromatography (hexane/ethyl acetate 9/1).

Yield: 3.88 g (59.6%).

¹ H NMR (CDCl₃) δ: 0.68 d,3H),1.04 (d,3H), 1.23-1.47 (m,2H), 1.63--1.9.(m,4H), 2.25 (m,1H), 3.38 (t,2H), 3.67 (s,3H), 3.68 (s,3H), 3.90-4.05(m,2H); ¹³ C NMR (CDCl₃) δ: 16.58, 19.03, 23.29, 31.75, 32.63, 33.13,33.58, 52.33, 55.13, 60.78, 163.60.

b)1-Azido-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-butane

1-Bromo-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]-butane(3.81 g, 11.95 mmol) were dissolved in 40 ml DMF and 3.25 g NaN₃ (50mmol) and 0.1 g NBu₄ I were added. The solution was kept between 70° and80° C. overnight and diluted with 50 ml water. After extraction withchloroform the organic layers were collected and dried (MgSO₄). Theresidue was purified by flash chromatography (hexane/ethyl acetate 9/1).

Yield: 2.17 g (64.6%).

¹ H NMR (CDCl₃) δ: 0.68 (d,3H), 1.04 (d,3H), 1.20-1.50 (m,2H), 1.53-1.91(m,4H), 2.26 (m,1H), 3.25 (t,2H), 3.68 (s,3H), 3.69 (s,3H), 3.90-4.07(m,2H). ¹³ C NMR (CDCl₃) δ: 16.58, 19.05, 21.83, 28.72, 31.76, 33.56,51.32, 52.35, 55.16, 60.79, 163.63.

c) Methyl(S)-2-amino-6-azidohexanoate

1-Azido-4-[(2R,5S)-3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]butane(2.17 g, 7.72 mmol) was dissolved in 30 ml dioxane and a solution of1.28 ml conc. HCl in 30 ml water (15.45 mmol) was added dropwise, themixture stirred under argon at ambient temperature overnight. Ammoniasolution was added until pH 9 was reached, the solution extracted withchloroform, dried (MgSO₄), and the valine methyl ester was removed bybulb-to-bulb distillation (0.05 torr, 25° C.).

Yield: 1.44 g (100%)

¹ H NMR (CDCl₃) δ: 1.40-1.87 (m,8H), 3.28 (t,2H), 3.43 (dd,1H), 3.73(s,3H). ¹³ C NMR (CDCl₃) δ: 22.93, 28.63, 34.36, 51.22, 51.98, 54.26,176.37.

d) (S)-2-Amino-6-azidohexanoic acid

Methyl-(S)-2-amino-6-azidohexanoate (1.44 g, 7.73 mmol) was dissolved in4 ml dioxane and cooled to 0° C. 3.87 ml 2N LiOH solution (7.74 mmol)was added, and the solution was stirred under argon overnight. TLCcontrol showed quantitative formation of (S)-2-amino-6-azidohexanoicacid. This crude solution was used in the next step.

e) (S)-2-(9-Fluorenylmethyloxycarbonylamino)-6-azidohexanoic acid

11.6 ml 1N NaHCO₃ solution (11.6 mmol) and a solution of 3.00 g9-fluorenyloxycarbonylchloride (11.6 mmol) in 12 ml dioxane were addedto the crude solution of (S)-2-amino-6-azidohexanoic acid (7.73 mmol)and stirring under argon was continued overnight. The solution wasacidified to pH 2 by addition of a KHSO₄ -solution, extracted with ethylacetate, dried (MgSO₄), and the residue was purified by flashchromatography (hexane/ethyl acetate/acetic acid 7/3/0.5).

Yield: 2.00 g=65.6%

¹ H NMR (DMSO) δ: 1.20-1.80 (m,2H), 3.31 (t,2H), 3.94 (m,1H), 4.27(m,3H), 7.20-8.00 (m,8H). ¹³ C NMR (DMSO) δ: 23.32, 28.26, 30.73, 47.10,50.94, 54.09, 66.01, 120.48, 125.65, 127.44, 128.02, 141.11, 144.19,144.25, 156.54, 174.22.

EXAMPLE 4 (S)-N-(9-Fluorenylmethyloxycarbonyl)-O-methyl-serine

a)(2R,5S)-3,6-Dimethoxy-5-(methoxymethyl)-2-isopropyl-2,5-dihydropyrazine

To a solution of (R)-2,5-dihydro-3,6-dimethoxy-2-isopropyl-pyrazine(5.53 g, 30 mmol) in anhydrous THF (80 ml) a 1.6M solution of n-BuLi inhexane (10.85 ml, 17.36 mmol) was added at -78° C. After 30 minutes asolution of bromomethyl methyl ether (4.5 g, 36 mmol) in THF (20 ml) wasadded dropwise. The reaction mixture was allowed to come to 20° C.overnight and was quenched by addition of 1M phosphate buffer (pH 7, 80ml). The turbid mixture was stirred for 10 minutes, diluted with water(250 ml) and diethyl ether (180 ml). The layers were separated and theaqueous phase extracted with diethyl ether (2×150 ml). The combinedorganic layer was dried (Na₂ SO₄), concentrated and purified by flashchromatography (silica gel; hexane:diethyl ether 5:1) to yield 5.61 g.To remove final impurities of the other diastereomer the material wascrystallized from acetonitrile at -30° C. and the cold precipitateredissolved in CH₂ Cl₂, dried (Na₂ SO₄) and concentrated.

Yield 4.24 g (18.6 mmol, 62%).

¹ H NMR (CDCl₃) δ: 4.05 (q, 1H, J 3.5 Hz), 3.99 (t, 1H, J 3.5 Hz),3.80-3.64 (m, 8H), 3.34 (s, 3H), 2.29 (dsept, 1H, J 3.5, 7.0 Hz), 1.05(d, 3H), 0.68 (d, 3H). ¹³ C NMR (CDCl₃) δ164.79, 161.58, 74.13, 60.76,59.51, 56.82, 52.42. FAB-MS signal at m/z 229 [M⁺ +1]. C₁₁ H₂₀ N₂ O₃(228.29).

b) Methyl(S)-2-(9-fluorenylmethyloxycarbonylamino)-3-methoxypropionate

To a solution of(2R,5S)-3,6-dimethoxy-5-(methoxymethyl)-2-isopropyl-2,5-dihydropyrazine(2.1 g, 9.2 mmol) in dioxane (18 ml ) 1M HCl (18.4 ml ), 18.4 mmol) wasadded and the mixture stirred at ambient temperature overnight. Thevolatile components were evaporated, the residue redissolved in dioxaneand again evaporated. The crude residue was dissolved dioxane and FmocCl(5.71 g, 2.21 mmol) was added. After 2 minutes 1M NaHCO₃ (44.2 ml, 44.2mmol) was added. After 15 minutes the mixture was diluted with water (50ml) and CHCl₃ (100 ml). The layers were separated and the aqueous phaseextracted with CHCl₃ (2×75 ml). The combined organic layer was dried(Na₂ SO₄), concentrated and purified by flash chromatography (silicagel; hexane: ethyl acetate 2:1).

Yield 2.68 g (7.54 mmol, 82%).

¹ H NMR (300 MHz, CDCl₃) δ7.78 (d,2H, J 7 Hz), 7.63 (t, 2H, J 7 Hz),7.41 (t, 2H, 7 Hz), 4.56-4.35 (m, 3H), 4.25 (t, 1H, J 3.0, 9.3 Hz), 3.84(dd, 1H, J 3.0, 9.3 Hz), 3.70 (s, 3H), 3.65 (dd, 1H, J 3.6, 9.3 Hz),3.36 (s, 3H). ¹³ C NMR (75 MHz, CDCl₃) δ170.77, 156.01, 143.88, 143.75,141.25, 127.66, 127.02, 125.12, 125.07, 119.92, 72.27, 67.11, 59.23,54.37, 53.44, 52.54, 47.12.

c) (S)-(9-Fluorenylmethyloxycarbonylamino)-3-methoxypropionic acid

To a solution of methyl(S)-(9-fluorenylmethyloxycarbonylamino)-3-methoxypropionate (2.18 g,6.13 mmol) in dioxane (31 ml) 6M HCl (31 ml, 184 mmol) was added. Thismixture was allowed to cool down and concentrated. The residue waspurified by flash chromatography (silica gel; hexane: ethyl acetate:acetic acid (10:10:1).

Yield: 1.91 g (5.60 mol, 91%) after lyophilization.

¹ H NMR (DMSO-d₆) δ7.87 (d, 2H, J 7.5 Hz), 7.74 (d, 2H, J 7.5 Hz), 7.41(dt, 2H, J 0.6 Hz, 7.5 Hz), 7.31 (dt, 2H, J 0.6 Hz, 7.5 Hz), 4.29-4.19(m, 4H), 3.65-3.54 (m, 2H), 3.24 (s, 3H). ¹³ C NMR (DMSO-d₆): δ172.07,156.46, 144.23, 144.21, 141.11, 128.03, 127.46, 125.73, 120.48, 71.80,66.19, 58.60, 54.47, 47.05.

EXAMPLE 5(S)-6-(Benzyloxycarbonylamino)-2,(9-fluorenylmethyloxy-carbonylamino)-hex-4-ynoicacid

a)1-Chloro-4-[(2R,5S)-3,6-diethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]but-2-yne:

To a solution of (R)-2,5-dihydro-3,6-diethoxy-2-isopropylpyrazine (21.23g, 100 mmol) in THF (200 ml) 1.6M n-BuLi (62.5 ml, 100 mmol) was addeddropwise at -78°C. After 30 minutes this solution was transferred to asolution of 1,4-dichlorobut-2-yne (49.2 g, 400 mmol) in THF (400 ml) at-78° C. The mixture was allowed to come to 20° C. overnight. Thereaction was quenched by addition of 1M phosphate buffer solution (250ml). The mixture was diluted with diethyl ether (300 ml) and water (300ml). The aqueous layer was separated and extracted with diethyl ether(2×300 ml). The combined organic layer was dried (Na₂ SO₄), filtered,concentrated and purified by flash chromatography (silica gel;hexane:diethyl ether 10:1).

Yield: 24.8 g (83 mmol, 83%).

¹ H NMR (CDCl₃): δ4.39-4.00 (m, 8H), 2.78 (m, AA'X, 2H), 2.30 (dsept,1H, J 3.5, 9.5 Hz), 1.38-1.20 (m, 6H), 1.05 (d, 3H, 7 Hz), 0.70 (d, 3H,7 Hz). ¹³ C NMR (CDCl₃): δ161.03, 159.25, 83.76, 60.90, 54.21, 31.68,30.95, 25.48, 20.62, 19.03, 15.85, 14.21.

b)1-Azido-4-[(2R,5S)-3,6-diethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]but-2-yne

A solution of1-chloro-4-[(2R,5S)-3,6-diethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]but-2-yne(19.4 g, 65 mmol) and tetrabutylammonium iodide (2 g) in anhydrous DMF(200 ml) was stirred overnight at 20° C. Most of the DMF was removed atreduced pressure (0.01 torr). The residue was partitioned betweendiethyl ether (250 ml) and water (250 ml). The aqueous layer extractedwith ether (2×250 ml). The combined organic layer was dried (Na₂ SO₄),concentrated and the residue purified by flash chromatography (silicagel; hexane:diethyl ether 8:1).

Yield: 18.06 g (59 mmol, 91%).

¹ H NMR (CDCl₃) δ4.25-3.99 (m, 4H), 3.84 (m, 6H), 3.84 (m, 2H),2.83-2.77 (m, AA'X, 2H), 2.33-2.26 (m, 1H), 1.35-1.27 (m, 6H), 1.05 (d,3H, J 7.2 Hz), 0.71 (d, 3H, J 7.2 Hz). ¹³ C NMR (CDCl₃) δ164.24, 161.16,84.54, 73.50, 61.00, 60.74, 54.31, 40.20, 31.84, 25.31, 19.03, 16.62,14.27, 14.21.

c)1-Benzyloxcarbonylamino-4-[(2R,5S)-3,6-diethoxy-2,5-dihydro-2-isopropyl-5-pyrazinyl]but-2-yne

To a solution of1-azido-4-[(2R,5S)-3,6-diethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl]but-2-yne(3.6 g, 11.8 mmol) in THF (236 ml) triphenylphosphine (3.7 g, 14.16mmol) and H₂ O (680 ml, 37.8 mmol) were added at 20° C. Immediate N₂-evolution was observed. The reaction mixture was stirred for 2 hoursand then concentrated. To a solution of the residue in dioxane (29 ml)1M NaHCO₃ (14.2 ml, 14.2 mmol) and Cbz-chloride (2.42 g, 14.2 mmol) wereadded at 0° C. The mixture was kept at this temperature for 1 hour, thenextracted with diethyl ether (3×50 ml). The combined organic layer wasdried (Na₂ SO₄), filtered, concentrated and purified by flashchromatography (silica gel; hexane:ethyl acetate 4:1).

Yield: 4.1 g (9.92 mmol, 84%).

¹ H NMR (CDCl₃): δ7.37-7.33 (m, 5H), 5.10 (s, 2H), 4.24-4.03 (m, 6H),3.97-3.90 (m, 4H), 2.70-2.67 (m, AA'X, 2H), 2.32-2.25 (m, 1H), 1.27 (t,4H, J 7 Hz), 1.04 (d, 3H, J 7 Hz), 0.69 (d, 3H, J 7 Hz). ¹³ C NMR(CDCl₃) δ164.11, 161.35, 136.31, 128.46, 128.42, 128.05, 127.53, 126.89,80.41, 66.85, 65.25, 60.85, 60.69, 60.65, 54.43, 31.66, 25.32, 19.054,16.53, 14.29, 14.21.

d) Ethyl (S)-2-Amino-6-(benzyloxycarbonylamino)-2-hexynoate

To a solution of1-benzyloxycarbonylamino-4-[(2R,5S)-3,6-diethoxy-2,5-dihydro-2-isopropyl-5-pyrazinyl]but-2-yne(4 g, 9.67 mmol) in dioxane (38 ml) 1M HCl (38.7 ml, 38.7 mmol) wasadded. This mixture was stirred at ambient temperature overnight. Mostof the dioxane was removed at reduced pressure and the aqueous residuediluted with water to 100 ml. This mixture was extracted with ether (100ml). Then the pH of the solution was adjusted to 9 by addition ofconcentrated NH₃ whereupon a white oil formed. This mixture wasextracted with CHCl₃ (3×50 ml), the combined CHCl₃ -- layer dried(MgSO₄), filtered and concentrated. The residue was subjected toKugelrohr distillation to remove the ethyl valinate at 50° C. (0.1torr). The remaining material was pure product.

Yield: 2.57 g (8.44 mmol, 87.3%).

¹ H NMR (CDCl₃): δ7.34 (s, 5H), 5.11 (s, 2H), 4.18 (q, 2H, J 7 Hz), 3.96(m, 2H), 3.57 (t, 1H, J 6 Hz), 2.60 (AA'X, 2H), 1.27 (t, 3H, J 7 Hz). ¹³C NMR (CDCl₃): δ173.73, 160.35, 136.24, 128.44, 128.10, 128.05, 79.02,78.66, 66.93, 61.19, 53.22, 31.15, 25.10, 14.15.

e)(S)-6-(Benzyloxycarbonylamino)-2-9-fluorenyl-methyloxycarbonylamino)-4-hexynoicacid

To a solution of ethyl(S)-2-amino-6-(benzyloxycarbonylamino)-4-hexynoate HCl (520 mg, 1.71mmol) in dioxane (5 ml) 1M NaHCO₃ (2.05 ml, 2.05 mmol) and water (3 ml).After 5 hours FmocCl (530 mg, 2.05 mmol) in dioxane (5 ml) was added.Almost immediately a precipitate formed. This mixture was acidified byaddition of 1M HCl and extracted with CHCl₃ (3×30 ml). The organic layerwas concentrated and purified by flash chromatography (silica gel;hexane:ethyl acetate:acetic acid 10:10:1).

Yield: 720 mg (1.44 mmol, 84.5%).

¹ H NMR (300 MHz, DMSO-d₆): δ7.87, (d, 2H, J 7.5 Hz), 7.71 (d, 2H, J 7.5Hz), 7.42-7.28 (m, 9H), 5.00 (s, 2H), 4.30-4.20 (m, 3H), 4.10 (m, 1H),3.77 (m, 2H), 2.58 (m, 2H). ¹³ C NMR (300 MHz, DMSO-d₆): δ172.41,156.28, 144.20, 141.10, 137.35, 128.73, 128.21, 128.03, 127.48, 125.68,120.49, 100.09, 79.37, 79.03, 66.21, 65.93, 53.49, 47.04, 30.63, 21.78.

EXAMPLE 6 (S)-2-Amino-3-(2-furyl) propionic acid

a) (2R,5S)-2.5-Dihydro-3,6dimethoxy-2-isopropyl-5-furfurylpyrazine

A 1.55M solution of butyllithium (6.45 ml, 10 mmol) in hexane was addedby syringe to a stirred solution of(2R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (1.84 g, 10 mmol) inTHF (50 ml) at -78° C. and the solution stirred for 1 hour at -78° C. Asolution of 2-chloromethylfuran (1.3 g, 11 mmol) in THF (10 ml) wasadded gradually and the stirring continued overnight. The solvent wasremoved under reduced pressure, the residue dissolved in ethyl acetate(50 ml) and extracted with phosphate buffer (pH 7). The water phase wasextracted twice with ethyl acetate (2×20 ml ). The organic layer wasdried (MgSO₄), solvent evaporated under reduced pressure and the residuepurified by flash chromatography (diethyl ether/hexane 15/1, silicagel).

Yield: 2.0 g (76%), colourless viscous liquid.

¹ H NMR (CDCl₃): δ0.65 (d, 3H, J 6.7 Hz), 1.00 (d, 3H, J 6.7 Hz), 2.20(m, 1H), 3.11 (d, 2H, J 5 Hz), 3.62 (t, 1H, J 3.5 Hz), 3.65 (d, 3H, J0.9 Hz), 3.70 (d, 3H, J 0.9 Hz), 4.26 (dd, 1H, J 5, J 8.5 Hz), 5.96 (d,1H, J 3.1 Hz), 6.23 (dd, 1H, J 1.8, J 3.1 Hz), 7.26 (dd, 1H, J 1.0, J0.8 Hz). ¹³ C NMR (CDCl₃): δ16.54, 19.07, 31.43, 33.03, 52.33, 52.41,54.95, 60.48, 107.10, 110.24, 141.28, 152.27, 162.53, 164.10.

b) Methyl (S)-2-amino-3-(2-furyl)propionate

A mixture of (2R,5S)-2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-furfurylpyrazine (2 g, 7.6mmol), 1M HCl (7.6 ml, 7.6 mmol) and dioxane (7.6 ml) was stirred atambient temperature overnight under N₂. Dioxane was removed underreduced pressure and the water phase extracted with diethyl ether (20ml). Aqueous ammonia was added to the water phase until pH 9, extractedwith CHCl₃ (3×30 ml ), dried (MgSO₄) and the solvent removed underreduced pressure. The valine methyl ester was removed by bulb-to-bulbdistillation at 40°-50° C. (0.05 mbar). The undistilled title compoundwas used without further purification.

Yield: 1.0 g (86%).

¹ H NMR (CDCl₃): δ1.5 (s, 2H), 2.92 (dd, 1H, J 15, J 7.2 Hz), 3.04 (dd,1H, J 15, J 5 Hz), 3.68 (d, 3H, J 0.5 Hz), 3.73 (dd, 1H, J 7.2, J 5.1Hz), 6.06 (dd, 1H, J 0.7, J 3.2 Hz), 6.24 (dd, 1H, J 2.9, J 2 Hz), 7.28(dd, 1H, J 0.7, J 1.9 Hz). ¹³ C NMR (CDCl₃): δ33.45, 52.00, 53.67,107.44, 110.20, 141.83, 151.32, 174.97.

c) (S)-2-(9-Fluorenylmethyloxycarbonylamino)-3-(2-furyl)-propionic acid

Methyl (S)-2-amino-3-(2-furyl)propionate (1 g, 6.5 mmol) was mixed with2M LiOH (3.27 ml, 6.5 mmol) and dioxane (3.27 ml ) at 0° C. and stirredovernight under N₂. The next day a TLC control (CHCl₃ /MeOH/NH₃ aq1/1/0.1) of the reaction mixture showed no ester present. 1M NaHCO₃(9.75 ml, 9.75 mmol) and 9-fluorenylmethyloxycarbonyl chloride (2.5 g,9.75 mmol) dissolved in dioxane (10 ml) were added to the above solutionand the stirring continued for a further 1 hour. Dioxane was removedunder reduced pressure and the aqueous solution acidified with a 10%KHSO₄ solution to pH 2. The solution was extracted with CHCl₃ (3×20 ml),dried (MgSO₄), evaporated and purified by flash chromatography(Hexane/ethyl acetate/acetic acid 10/10/1).

Yield: 1.3 g (53%), white crystals.

¹ H NMR (CDCl₃): δ2.95 (dd, 1H, J 9.9, J 15.2 Hz), 3.08 (dd, 1H, J 4.4,J 15.2 Hz), 3.6 (br.s, 1H), 4.16-4.26 (m, 4H), 6.12 (d, 1H, J 2.9 Hz),6.33 (t, 1H, J 2.3 Hz), 7.29 (t, 2H, J 7.32 Hz), 7.39 (t, 2H, J 7.48Hz), 7.49 (br.s, 1H), 7.64 (dd, 2H, J 7.17, J 2.14 Hz), 7.85 (d, 2H, J7.48 Hz). ¹³ C NMR (CDCl₃): δ29.8, 46.98, 66.04, 107.45, 110.85, 120.48,125.57, 125.63, 127.50, 128.07, 141.06, 142.21, 144.09, 151.84, 156.26,160.35.

EXAMPLE 7 (S)-2-Amino-3-(3,5-dimethyl-4-isoxazolyl)propionic acid

a)(2R,5S)-2,5-Dihydro-3,6-dimethoxy-2-isopropyl-5-(3,5-dimethyl-4-isoxazolylmethyl)pyrazine

A 1.55M solution of butyllithium (10 ml, 15.5 mmol) in hexane was addedby syringe to a stirred solution of(2R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (2.85 g, 15.5 mmol)in THF (50 ml) at -78° C. and the solution stirred for 1 hour at -78° C.A solution of 4- chloromethyl-3,5-dimethylisoxazole (2.34 g, 16 mmol) inTHF (10 ml) was added gradually and the stirring continued overnight.The solvent was removed under reduced pressure, the residue dissolved inethyl acetate (50 ml) and extracted with phosphate buffer (pH 7). Thewater phase was extracted twice with ethyl acetate (2×20 ml). Theorganic layer was dried (MgSO₄), solvent evaporated under reducedpressure and the residue purified by flash chromatography (diethylether/hexane 4/1, silica gel).

Yield: 3 g (64%), colourless viscous liquid.

¹ H NMR (CDCl₃): δ0.60 (d, 3H, J 6.7 Hz), 0.97 (d, 3H, J 6.7 Hz), 2.17(s, 3H), 2.26 (s, 3H), 2.73 (dd, 1H, J 5.9, J 14.6 Hz), 2.86 (dd, 1H, J4.4, J 14.7 Hz), 3.59 (t, 1H, J 3.4 Hz), 3.63 (d, 3H, J 0.3 Hz), 3.68(d, 3H, J 0.3 Hz), 4.11 (m, 1H). ¹³ C NMR (CDCl₃): δ10.21, 11.16, 16.41,18.99, 26.99, 31.29, 52.26, 52.33, 55.93, 60.46, 109.82, 160.18, 162.33,164.24, 166.34.

b) Methyl (S)-2-amino-3-(3,5-dimethyl-4-isoxazolyl)propionate

A mixture of(2R,5S)-2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-(3,5-dimethyl-4-isoxazolylmethyl)pyrazine(2 g, 6.8 mmol), 1.0M HCl (13.6 ml, 13.6 mmol) and dioxane (13.6 ml) wasstirred at ambient temperature overnight under N₂. Dioxane was removedunder reduced pressure and the water phase extracted with diethyl ether(20 ml). Aqueous ammonia was added to the water phase until pH 9,extracted with CHCl₃ (3×30 ml), dried (MgSO₄) and the solvent removedunder reduced pressure. The valine methyl ester was removed bybulb-to-bulb distillation at 40°-50° C. (0.05 mbar). The undistilledtitle compound was used without further purification.

Yield: 1.3 g.

¹ H NMR (CDCl₃): δ1.52 (br.s, 2H), 2.21 (s, 3H), 2.31 (s, 3H), 2.56 (dd,1H, J 7.5, J 14.6 Hz), 2.74 (dd, 1H, J 6.2, J 14.6 Hz), 3.55 (t, 1H, J6.7 Hz), 3.69 (s, 3H).

¹³ C NMR (CDCl₃): δ0.18, 11.06, 27.94, 52.06, 54.45, 109.45, 159.63,166.31, 175.18.

c) (S)-2-(9-Fluorenylmethyloxycarbonylamino)-3-(3,5-dimethyl-4-isoxazolyl)propionicacid

Methyl (S)-2-amino-3-(3,5-dimethyl-4-isoxazolyl)propionate (1.3 g, 6.6mmol) was mixed with 2M LiOH (3.3 ml, 6.6 mmol) and dioxane (3.3 ml) at0° C. and stirred overnight under N₂. The next day a TLC control (CHCl₃/MeOH/NH₃ aq 1/1/0.1) of the reaction mixture showed no ester present.1M NaHCO₃ (10 ml, 10 mmol) and 9-fluorenylmethyloxycarbonyl chloride(2.6 g, 10 mmol) dissolved in dioxane (10 ml) were added to the abovesolution and the stirring continued for a further hour. Dioxane wasremoved under reduced pressure and the aqueous solution acidified with a10% KHSO₄ solution to pH 2. The solution was extracted with CHCl₃ (3×20ml), dried (MgSO₄) evaporated and purified by flash chromatography(Hexane/ethyl acetate/acetic acid 10/10/1).

Yield: 1.6 g (59%), white crystals.

¹ H NMR (CDCl₃): δ2.10 (s, 3H), 2.19 (s, 3H), 2.65 (dd, 1H, J 9.46, J14.65 Hz), 2.76 (dd, 1H, J 5.4, J 14.65 Hz), 3.5-4.2 (m, 4H), 7.24-7.40(m, 4H), 7.63 (d, 2H, J 7.32 Hz), 7.82 (t, 2H, J 7.63 Hz). ¹³ C NMR(CDCl₃): δ10.02, 10.90, 24.27, 46.94, 53.81, 66.73, 110.16, 120.44,125.51, 127.46, 128.06, 141.06, 144.12, 156.27, 159.83, 166.51, 173.02.

Solid-phase synthesis of peptides

Solid-phase peptide synthesis was carried out essentially according tothe principles of the fluorenylmethoxycarbonyl (Fmoc)-polyamide strategy(Atherton & Sheppard, Solid phase peptide synthesis: a practicalapproach. Oxford: IRL Press at Oxford University Press, 1989).Commercially available synthesis resins were used; for batch synthesiseither manually or using a semi-automatic instrument (Labortec PeptideSynthesizer 5P 650) these were of polystyrene with acid-labile (Wang, J.Am, Chem. Soc., 95, 1328-1333, 1973) or acid hyperlabile linkage agents(Merger et al., Tetrahedron Letters 29, 4005-4008, 1988). Alternatively,peptides were assembled in fully automatic mode on flow resins (Athertonet al., J. Chem. Soc., Chem. Commun., 1151-2, 1981) using an LKB Biolynx4170 Automated Peptide Synthesizer. Synthesis resins were purchasedwhich already contained the protected desired C-terminal Fmoc-amino acidresidue. Chain elongation was achieved variously with side-chainprotected Fmoc-amino acid pentafluorophenyl esters (Kisfaludy & Schoen,Synthesis, 325-327, 1983), using activation withdicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) (Koenig &Geiger, Chem. Ber. 103, 2034-2040, 1970) or using the coupling reagentPyBOP (Coste et al., Tetrahedron Lett., 31, 205-208, 1990). The lysineside-chain amino group was protected with the t-butyloxycarbonylfunction, the side-chain carboxy groups of glutamic and aspartic acidwere protected as the t-butyl esters.

After complete solid-phase assembly of the desired sequences, thepeptides were cleaved from the synthesis resins with concomitantside-chain deprotection using trifluoroacetic acid to which suitablescavenger chemicals (King et al., Int. J. Peptide Protein RES., 36255-268, 1990) had been added. After evaporation, the peptides wereisolated by precipitation with diethyl ether and drying. Purificationwas by preparative reversed phase high performance liquidchromatography.

                  TABLE 1                                                         ______________________________________                                        Analytical data for monomeric peptides                                        with the general structure                                                    Pyr--Glu--Asp--Xaa--Lys--OH SEQ ID NO 3                                                      HPLC Method.sup.b                                                             (Retention  Purity.sup.c                                                                          FAB-MS                                     Xaa.sup.a      time, min)  (%)     [M + H].sup.+                              ______________________________________                                        Valine         0-30-20 (17)                                                                              100     601.4                                      Norleucine     0-30-20 (23)                                                                              99      615.4                                      Leucine        0-30-20 (22)                                                                              98      615.4                                      Tryptophan     20-50-20 (14)                                                                             96      688.3                                      Tyrosine       0-30-20 (13)                                                                              98                                                 Lysine         0-30-20 (10)                                                                              100     630.4                                      Glutamic acid  0-30-20 (12)                                                                              99      631.6                                      Glutamine      0-30-20 (12)                                                                              100     630.3                                      Histidine      0-30-20 (13)                                                                              99      639.4                                      Proline        0-30-20 (15)                                                                              99      599.3                                      1-Naphthylalanine                                                                            30-90-20 (15)                                                                             99      699                                        Thien-2-ylalanine                                                                            0-30-20 (20)                                                                              100     655.3                                      Pyridin-2-ylalanine                                                                          0-30-20 (12)                                                                              95                                                 Furan-2-ylalanine                                                             3,5-Dimethylisoxazol-4-                                                       ylalanine                                                                     Cyclohexylalanine                                                                            10-40-20 (23)                                                                             98      655.4                                      Allylglycine                                                                  O-Methylserine                                                                2-Amino-6-azidohexanoic                                                       acid                                                                          2-Amino-4,5-dihydroxy-6-                                                      benzyloxycarbonylamino-                                                       hexanoic acid                                                                 ______________________________________                                         .sup.a All amino acids have the L configuration, i.e., that found in          natural amino acids                                                           .sup.b The methods are expressed as gradients of mobile phase B in A over     time, thus e.g. 1040-20 refers to a gradient starting at 10 and finishing     at 40% B over 20 min. Mobile phases: A) 0.1% TFA B) 0.1% TFA in 40% MeCN.     Column: Vydac TP54,C18,0.46 × 25 cm, 5 μm particles, 100 Å       pore; flow 1 mL/min                                                           .sup.c Refers to integration of HPLC chromatogram peaks (λ = 215       nm)                                                                      

                                      TABLE 2                                     __________________________________________________________________________    Amino acid analysis data for peptides                                         Peptide with Xaa in Pyr--Glu--Asp--Xaa--Lys--OH SEQ ID NO 4                   Xaa═Val                                                                              Xaa═Leu                                                                          Xaa═Nle                                                                          Xaa═Ser                                                                         Xaa═Trp                                    Number of residues found (theory)                                             __________________________________________________________________________    Val 0.81(1)                                                                   Nle               0.94(1)                                                     Leu        0.97(1)                                                            Trp                            0.51.sup.a (1)                                 Lys 0.77(1)                                                                              0.68(1)                                                                              0.89(1)                                                                              0.90(1)                                                                             1.00(1)                                        Glu 2.09(2)                                                                              2.01(2)                                                                              2.11(2)                                                                              2.11(2)                                                                             2.33(2)                                        Asp 1.00(1)                                                                              1.00(1)                                                                              1.00(1)                                                                              1.00(1)                                                                             0.68(1)                                        __________________________________________________________________________     .sup.a Tryptophan is partly destroyed during acid hydrolysis.            

HOBt=Hydroxybenzotriazole

Pfp=Pentafluorophenyl

Fmoc=9-Fluorenylmethoxycarbonyl

Boc=t-Butoxycarbonyl

DCC=Dicyclohexylcarbodiimide

Cin=(S)-2-Amino-5-phenyl-4-(E)-penten-1-oic acid.

BuLi=n-butyllithium

THF=tetrahydrofuran

DMF=dimethylformamide

pGlu-Glu-Asp-[Cin]-Lys SEQ ID NO 4

The peptide is synthesized using a Labortec Peptide Synthesizer.Fmoc-Lys(Boc)-Sasrin polymer (1.0 g, 0.6 mmol; Bachem A. G.;substitution 0.6 mmol/g) is charged into a 100 ml reaction flask.(S)-2-(9-Fluorenylmethoxycarbonylamino)-5-phenyl-4-(E)-penten-1-oic acid(578 mg, 1.4 mmol), DCC (290 mg, 1.4 mmol) and HOBt (211 mg, 1.4 mmol)in DMF (20 ml) are added to the polymer and the reaction allowed toproceed for 9 hours. The polymer is then washed with CH₂ Cl₂, with 30%MeOH in CH₂ Cl₂ and with DMF (negative Kaiser test).

The remaining part of the synthesis is carried out by standard protocolusing Fmoc-Asp(OtBu)-Opfp (0.89 g, 1.5 mmol), Fmoc-Glu(OtBu)-Opfp (0.90g, 1.5 mmol) and pGlu-pentachlorophenyl ester (0.57 g, 1.5 mmol). HOBt(0.23 mg, 1.5 mmol) is added in each coupling step which is allowed toproceed for 1 hour. Completion of the coupling is ascertained bynegative Kaiser test. After the coupling with the Fmoc-amino acid thepolymer is washed with DMF, the protecting group cleaved off by 20%piperidine in DMF, and the polymer again washed with DMF. After thefinal coupling the polymer is washed with MeOH/CH₂ Cl₂ and CH₂ Cl₂ anddried. The peptide is cleaved from the polymer by TFA:CH₂ Cl₂ 1:1, thesolution freeze-dried, the residue dissolved in water, filtered (0.45 μ)and the filtrate freeze-dried. For further purification the crudepeptide is dissolved in water and subjected to preparative HPLC onBeckman Ultrasphere ODS and solutions A0.1% TFA in H₂ O, B 0.1% TFA inMeCN:H₂ O 40:60. The pure peptide (>95%) is obtained after freeze-dryingof the fractions collected which contain the homogenous peptide.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label=pE                                              /note="pE=pyroglutamic acid"                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      XaaGluAspCysLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      LysIleIleHisGluAspGlyTyrSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label=pE                                              /note="pE=pyroglutamic acid"                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      XaaGluAspXaaLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /label=Cin /note="Cin=(S)-2-amino-5                    phenyl-4-(E)-penten-1-oic acid"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GluGluAspXaaLys                                                               15                                                                            __________________________________________________________________________

We claim:
 1. A peptide compound derivative comprising a single chainhemoregulatory peptide of formula: ##STR6## wherein R^(a) represents##STR7## and A is a group --CR^(A) R^(A') --Z, wherein n and mindependently represent 0 or 1;q and r independently represent 1 or 2; srepresents 3 or 4; R¹ and R² are both hydrogen atoms or, when m=0, R¹and R² together represent an oxo group; R³ and R⁴ are both hydrogenatoms or together represent a carbon-carbon bond; each R⁶ and R⁷independently represents a hydroxy group or an amino group; R⁹represents hydrogen or a methyl group; and R¹⁰ represents a hydroxy oramino group, the residue of the amino acid glutamine or a peptide havingan N-terminal glutamine unit, each R^(A) is independently a hydrogenatom or a group --R^(A"), --OR^(A"), --SR^(A"), --NR^(A") R^(A"),CONR^(A") R^(A") or --COOR^(A") ; R^(A') is a hydrogen atom or a groupR^(A") ; R^(A") is an alkyl, cycloalkyl, alkanoyl, hydroxyalkyl, amidinegroup or a carbocyclic or heterocyclic group; Z is a group --OR^(B),--NR^(C) R^(C), --CR^(D) R^(E) R^(F) or a substituted or unsubstituted,aromatic or non-aromatic, carbocyclic or heterocyclic ring other thanunsubstituted phenyl; each R^(B) is a straight chained or branched,saturated or unsaturated hydrocarbon group optionally substituted byhydroxyl, amino or azide groups or by one or more R^(A) groups, whereR^(A) is as defined above, and optionally interrupted by one or more--N--, --O-- or --S-- heteroatoms; R^(C) is a hydrogen atom or a groupR^(B) ; R^(D) is a hydrogen atom or a group R^(F) ; R^(E) is a hydrogenatom or a group R^(F), or together with the carbon atom of the CR^(D)R^(E) R^(F) group, R^(E) and R^(D) form a>C═O group; and R^(F) is agroup --R^(B), --OR^(B), --NR^(C) R^(C) or --SR^(B), or a hydroxy,carboxy, aminocarbonyl or alkoxy group, or is a methylene group linkedto the nitrogen atom of the --NH--CH (A)--CO--group, or together withR^(D) forms an alkylidene group, or is a hydrogen atom where one ofR^(A), R^(A'), R^(D) and R^(E) is other than hydrogen.
 2. A peptidecompound derivative as claimed in claim 1 wherein --A is a group

    --CH.sub.2 --Z

wherein Z is a group --OR^(B), where R^(B) is as defined in claim 1 or--CHR^(D) R^(F) ; R^(D) represents a hydrogen atom or a group --CH₃ ;R^(F) represents an alkyl group optionally substituted by hydroxyl,amino or azide groups; or R^(D) and R^(F) together form an alkylidinegroup.
 3. A peptide compound derivative as claimed in claim 1 wherein ina peptide chain of formula I n represents
 0. 4. A peptide compoundderivative as claimed in claim 1 wherein in a peptide chain of formula Im represents
 0. 5. A pharmaceutical composition comprising a peptidecompound derivative as claimed in claim 1 together with a pharmaceuticalcarrier or excipient.
 6. A method of inhibiting cell division in apatient, said method comprising administering to said patient aneffective amount of a peptide compound derivative as claimed in claim 1.7. A method as claimed in claim 6 wherein division of myelopoietic orbone marrow cells is inhibited.
 8. A process for producing a peptidecompound derivative as claimed in claim 1 comprising deprotecting apartially or fully protected derivative thereof.
 9. Lactim dipeptideethers of formula ##STR8## wherein --A is as defined in claim 1 and whenR^(A) and R^(A') each represent hydrogen then Z is other than asubstituted aromatic carbocyclic or heterocyclic ring.
 10. Aminoprotected, hydroxyl protected, thiol protected and/or carboxyl protectedderivatives of a peptide compound derivative comprising a single-chainhemoregulatory peptide of formula: ##STR9## wherein ##STR10## and A is agroup --CR^(A) R^(A') --Z, wherein n and m independently represent 0 or1;q or r independently represent 1 or 2; s represents 3 or 4; R¹ and R²are both hydrogen atoms or, when m=0, R¹ and R² together represent anoxo group; R³ and R⁴ are both hydrogen atoms or together represent acarbon-carbon bond; each R⁶ and R⁷ independently represents a hydroxygroup or an amino group; R⁹ represents hydrogen or a methyl group; andR¹⁰ represents a hydroxy or amino group, the residue of the amino acidglutamine or a peptide having an N-terminal glutamine unit, each R^(A)is independently a hydrogen atom or a group --R^(A"), --OR^(A"),--SR^(A"), --NR^(A") R^(A"), CONR^(A") R^(A") or --COOR^(A") ; R^(A') isa hydrogen atom or a group R^(A") ; R^(A") is an alkyl, cycloalkyl,alkanoyl, hydroxyalkyl, amidine group or a carbocyclic or heterocyclicgroup; Z is a group --OR^(B), --NR^(C) R^(C), --CR^(D) R^(E) R^(F) or asubstituted or unsubstituted, aromatic or non-aromatic, carbocyclic orheterocyclic ring other than unsubstituted phenyl; each R^(B) is astraight chained or branched, saturated or unsaturated hydrocarbon groupoptionally substituted by hydroxyl, amino or azide groups or by one ormore R^(A) groups, where R^(A) is as defined above, and optionallyinterrupted by one or more --N--, --O-- or --S-- heteroatoms; R^(C) is ahydrogen atom or a group R^(B) ; R^(D) is a hydrogen atom or a groupR^(F) ; R^(E) is a hydrogen atom or a group R^(F), or together with thecarbon atom of the CR^(D) R^(E) R^(F) group, R^(E) and R^(D) form a>C═Ogroup; and R^(F) is a group --R^(B), --OR^(B), --NR^(C) R^(C) or--SR^(B), or a hydroxy, carboxy, aminocarbonyl or alkoxy group, or is amethylene group linked to the nitrogen atom of the --NH--CH(A)--CO--group, or together or with R^(D) forms an alkylidene group, or is ahydrogen atom where one of R^(A), R^(A'), R^(D) and R^(E) is other thanhydrogen.
 11. A peptide compound derivative comprising a single chainhemoregulatory peptide of formula: ##STR11## wherein ##STR12## and A isa group --CR^(A) R^(A') --Z, wherein n and m independently represent 0or 1;q and r independently represent 1 or 2; s represents 3 or 4; R¹ andR² are both hydrogen atoms or, when m=0, R¹ and R² together represent anoxo group; R³ and R⁴ are both hydrogen atoms or together represent acarbon-carbon bond; each R⁶ and R⁷ independently represents a hydroxygroup or an amino group; R⁹ represents hydrogen or a methyl group; andR¹⁰ represents a hydroxy or amino group, the residue of the amino acidglutamine or a peptide having an N-terminal glutamine unit, each R^(A)is independently a hydrogen atom or a group --R^(A"), --OR^(A"),--SR^(A"), --NR^(A") R^(A"), CONR^(A") R^(A") or --COOR ^(A") ; R^(A')is a hydrogen atom or a group R^(A") ; R^(A") is an alkyl, cycloalkyl,alkanoyl, hydroxyalkyl, amidine group or a carbocyclic or heterocyclicgroup; Z is a substituted or unsubstituted, aromatic or non-aromatic,carbocyclic or heterocyclic ring other than unsubstituted phenyl.
 12. Apeptide compound derivative as claimed in claim 11 wherein --A is

    --CH.sub.2 --Z

wherein Z represents a substituted or unsubstituted, aromatic ornon-aromatic, carbocyclic or heterocyclic ring other than unsubstitutedphenyl.
 13. A peptide compound derivative as claimed in claim 12 whereinZ represents a benzyl, indolyl, hydroxyphenyl, imidazolyl, naphthyl,thienyl, pyridinyl, furanyl, isoxazolyl, 3,5-dimethylisoxazolyl orcyclohexyl group.
 14. A peptide compound of formula ##STR13## wherein Ais a group --CR^(A) R^(A') --Z;in which R^(A) is a hydrogen atom or agroup --R^(A"), --OR^(A"), --SR^(A"), --NR^(A") R^(A"), CONR^(A") R^(A")or --COOR^(A") ; R^(A') is a hydrogen atom or a group R^(A") ; R^(A") isan alkyl, cycloalkyl, alkanoyl, hydroxyalkyl, amidine group or acarbocyclic or heterocyclic group; Z is a group --OR^(B), --NR^(C)R^(C), --CR^(D) R^(E) R^(F) or a substituted or unsubstituted, aromaticor non-aromatic, carbocyclic or heterocyclic ring other thanunsubstituted phenyl; wherein each R^(B) is a straight chained orbranched, saturated or unsaturated hydrocarbon group optionallysubstituted by hydroxyl, amino or azide groups or by one or more R^(A)groups, where R^(A) is as defined above, and optionally interrupted byone or more --N--, --O-- or --S-- heteroatoms; R^(C) is a hydrogen atomor a group R^(B) ; R^(D) is a hydrogen atom or a group R^(F) ; R^(E) isa hydrogen atom or a group R^(F), or together with the carbon atom ofthe CR^(D) R^(E) R^(F) group, R^(E) and R^(D) form a>C═O group; andR^(F) is a group --R^(B), --OR^(B), --NR^(C) R^(C) or --SR^(B), or ahydroxy, carboxy, aminocarbonyl or alkoxy group, or is a methylene grouplinked to the nitrogen atom of the --NH--CH(A)--CO-- group, or togetherwith R^(D) forms an alkylidene group, or is a hydrogen atom where one ofR^(A), R^(A'), R^(D) and R^(E) is other than hydrogen.